This study describes the in vivo activity of fexinidazole against Trypanosoma cruzi, the protozoan parasite causing Chagas disease, using mice infected with parasite strains with varying susceptibility to benznidazole, the standard treatment for Chagas. Fexinidazole and benznidazole were shown to have similar activity against benznidazolesusceptible and partially resistant T. cruzi strains (CL and Y), but fexinidazole had potent activity against benznidazole-resistant strains (VL-10 and Colombian). Fexinidazole treatment resulted in parasitological cure during acute disease phase in 88.9% of mice infected with the VL-10 strain and 78% with Colombian strain; benznidazole treatment did not result in cure in animals infected with these strains. Fexinidazole treatment was also shown to reduce myocarditis in all VL-10- and Colombian-infected animals, although parasite eradication was not achieved in all treated animals. These data demonstrate that it is possible to achieved better cure rates with fexinidazole in these experimental infection models than what is achieved with the standard benznidazole at the doses tested in this animal study benznidazole treatment regimen.